RU2685505C1 - Tyrosine heterocyclic amides, having analgesic, anti-inflammatory and antiedemic action - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention discloses heterocyclic derivatives of tyrosine of general formula (I) having anti-inflammatory, analgesic and antiedemic action.

(I),

.

EFFECT: disclosed derivatives can be used for cosmetic and therapeutic purposes in creams, ointments or gels, having analgesic, anti-inflammatory and antiedemic action, which do not cause side effects during external use.

1 cl, 6 dwg, 7 tbl, 2 ex

Description

Heterocyclic tyrosine derivatives with analgesic, anti-inflammatory and anti-edema effects

The invention relates to physiologically active derivatives of tyrosine, with analgesic, anti-inflammatory and anti-edema action. The inventive derivatives can be used in cosmetic and therapeutic purposes in the composition of creams, ointments or gels with a specified spectrum of activity.

There are a number of medical and cosmetic products, which include nonsteroidal anti-inflammatory agents with analgesic and anti-inflammatory properties. These are small molecules of various chemical structures, the most important of them for outdoor use:

1) salicylic acid derivatives, for example, in combination with betamethasone dipropionate (RF Patent No. 2223465, IPC А61К 31/573, publ. February 20, 2004);

2) derivatives of indole acetic acid, namely indomethacin in the composition of the ointment "Indomethacin" (Instructions for use, Indomethacin ointment 10%, manufacturer Biosynthesis, Russia) and gel "Indovazin" (Instructions for use, Indovazin gel, 45 g);

3) derivatives of phenylacetic acid, namely diclofenac in the composition of the preparations Voltaren Emulgel (Instructions for use, Voltaren emulgel, gel), Diklovit, etc. (Instructions for use, Diklovit gel 1%);

4) propionic acid derivatives, namely ibuprofen - "Nurofen gel" (Instructions for use Nurofen gel, 5%), ketoprofen - "Fastum gel" (Instructions for use Fastum gel) and "Ketonal gel" (Instructions for use Ketonal gel) ;

5) oxicamers, namely piroxicam - "Piroxicam-gel" (Instructions for use Piroxicam gel), "Finalgel" (Instructions for use FinalGel);

6) sulfonamide derivatives, namely nimesulide - “Naiz Gel” (Instructions for use of Naiz Gel)

These substances are part of various ointments, gels and creams, which are used as painkillers and anti-inflammatories, mainly in the treatment of acute and chronic inflammatory diseases of the musculoskeletal system, as well as sports injuries (bruises, damage to ligaments and muscles). A well-known disadvantage of nonsteroidal anti-inflammatory drugs is direct or indirect damage to the gastrointestinal mucosa (GIT), especially with prolonged use (diarrhea, gastric and duodenal ulcer, gastrointestinal bleeding), as well as the risk of developing cardiovascular complications (strokes, myocardial infarction). , peripheral arterial thrombosis). In addition, if these agents are applied over large areas for a long time and in high doses, systemic side effects may occur.

Also known nonsteroidal anti-inflammatory drugs based on the physiologically active components of the poisons of bees and snakes.

Bee venom is a complex mixture of biologically active substances. It contains enzymes, biogenic amines, amino acids (tryptophan), peptides - mellitin, apamine, as well as choline, free fatty acids, sugars, lipids, free bases, organic acids (phosphoric), magnesium, and trace elements. Peptides make up more than 50% of the dry weight of bee venom and account for most of its physiological activity. The best known remedy based on bee venom is Apizartron ointment (Instructions for use of Apizartron, ointment).

Cobra poisons and vipers have a similar anti-inflammatory and analgesic activity. Snake venom contains amino acids and proteins (including enzymes); fatty acids, trace elements. Ointment "Viprosal", Kobratoks, etc.

Preparations based on poisons of bees and snakes are used in low doses and do not have such side effects as the compounds listed above, however poisons of bees and snakes are strong allergens and can cause an allergic reaction in a patient. In addition, the raw materials for such ointments is difficult and expensive. This raw material is difficult to standardize, which is very important for industrial production.

A known group of neuropeptides, which are endogenous ligands - opioid receptor agonists. These peptides have an analgesic effect. Endogenous opioid peptides include endorphins, enkephalins, dinorphins, etc. The brain opioid peptide system plays an important role in the formation of motivations, emotions, behavioral affections, responses to stress and pain, and in the control of food intake. Peptides, as a rule, are non-toxic, act in low doses, are not addictive, there are enzyme systems in the body for their degradation to amino acids. Low molecular weight peptides and amino acid derivatives do not possess antigenic and immunogenic properties. Peptide substances are available and easy to standardize.

A specific feature of the structure of neuropeptides is the presence of tyrosine in their composition, which is a functionally important amino acid. Tyrosine itself also has physiological activity, but its use as a drug is limited due to the low solubility of this amino acid in aqueous media.

The objective of this invention is the development of tyrosine derivatives for use in cosmetic or medicinal creams, gels or ointments with analgesic, anti-inflammatory and anti-edematous action, not causing side effects during external use by the gastrointestinal tract, cardiovascular system, allergic reactions and addiction.

The technical result of the invention is to achieve analgesic, anti-inflammatory and anti-edema effects without side effects, allergic reactions and addiction.

The technical result is achieved by synthesizing tyrosine derivatives of the formula H - Tyr - X, where X is the group that provides the solubility of the compound in aqueous media.

The carbon atom in the five-membered heterocycle, marked with an asterisk, is optically active and can have either the L or D configuration.

The non-obviousness of the invention is due to the fact that any design of organic molecules based on amino acids is a purely theoretical approach. In the series of amino acid derivatives or their analogues, there is no clear structure-activity relationship, since each modified amino acid derivative is a unique chemical compound, its biological activity is not obvious and requires experimental confirmation.

Synthesis of the claimed compounds of formula (I) is carried out by peptide chemistry in solution by condensation of the corresponding heterocyclic imino acid (oxazolidicarboxylic - for derivatives (Ia) or thiazolidinecarboxylic - for compounds (Ib)), obtained by the described methods, with activated di-Boc-L-ester tyrosine with the subsequent removal of the protective groups to the compounds of formula (I). For the purification of the compounds of formula (I), reprecipitation and crystallization were used, and, if necessary, ion-exchange chromatography on cation-exchange resin.

The structure of the obtained compounds was confirmed by ¹ H NMR spectroscopy, and their homogeneity by high performance liquid chromatography (HPLC) data.

The anti-inflammatory and analgesic effects of the claimed tyrosine derivatives were studied in an acute experiment on a model of formalin paw edema in mice and in a subchronic experiment on a model of adjuvant arthritis in rats. At the end of the subchronic experiment, a withdrawal syndrome was evaluated. The claimed compounds for studying biological activity were introduced into the composition of a neutral cosmetic cream (cream A contained the claimed compound of formula (Ia), and cream B contained the claimed compound of formula (Ib)). The acute inflammatory reaction was reproduced by subplantar introduction of a 2% formalin solution into the right hind paw. Creams containing the claimed derivatives of tyrosine (hereinafter referred to as “Cream A”, “Cream B”) were applied to the paw 2 and 1 hour before the introduction of formalin. The anti-inflammatory effect of “Creams A and B” was evaluated by the severity of edema, the analgesic action by the behavior of animals (in points), by motor activity (in the open field test).

The results of the study showed that when using the cutaneous application of the proposed cosmetic products - “Cream A” and “Cream B”, the pain sensitivity decreases and the severity of formalin-induced paw edema decreases. The depression of edema averaged about 10%.

Chronic inflammation (adjuvant arthritis) was modeled on female rats by subplantar administration of 0.1 ml of Freund's complete adjuvant (PAF) into the right hind paw (Becton, Dickinson and Company, U.S.A. Lot 6292862). The control cream and the claimed cosmetics were applied to the rats one day after the administration of PAF and then daily for 27 days. The inflammatory response was evaluated in dynamics every 2-3 days by the intensity of inflammatory changes in the joints of the affected limb, expressed as a percentage of edema, as well as by changes in pain sensitivity thresholds. The primary reaction (edema on the injection paw) was evaluated on the 3rd day after the injection of the adjuvant.

On the second day after the withdrawal of the cream, the animals were tested for the presence of withdrawal syndrome. The abolition syndrome was evaluated on the second day after the abolition of the claimed cosmetic products - "Cream A" and "Cream B", registering analgesic activity, physical activity in the open field test, the general condition of impaired ear and corneal reflexes, the presence of seizures, tremor, ptosis , jumping, shaking, knocking teeth, scratching, sneezing, lateral position.

In the process of a subchronic experiment, it was found that the use of "Creams A and B" after creating an adjuvant arthritis inhibits the development of the inflammatory process. The maximum inhibition of edema was 20–25% for days 7–15 of the study for Crema B.

After discontinuation of the use of the claimed cosmetic products, no withdrawal syndrome was detected. The behavior and reflexes of animals have not changed.

The obtained results testify to the high effectiveness of the proposed "Cream B" as an anti-inflammatory and analgesic.

Example 1. Obtaining N-tyrosyl-4-carbonyl-5-methyl-1,3-oxosolidine (Ia)

To a suspension of 1.95 g (15.0 mmol) of 4-carboxy-5-methyl-1,3-oxosolidine [M.R. Vishnumaya, VK Singh Highly efficient reaction in organic and aqueous media // J.Org. Chem. 2009, v. 74, p. 4289-4297] in 40 ml of dry dimethylformamide (DMF) with stirring, 4.1 ml (29.3 mmol) of triethylamine and 7.2 g (15.0 mmol) of Boc ₂ -Tyr-ONSu were added and stirred at room temperature for 15 hours. The solvents were evaporated, the residue was dissolved in 50 ml of ethyl acetate and successively washed with 5% solution of sulfuric acid and water until neutral pH, the ethyl acetate layer was evaporated. The residue was dissolved in 20 ml of methylene chloride and 16.5 ml of trifluoroacetic acid was added. The reaction mixture was kept at room temperature for 1 hour and the trifluoroacetic acid was evaporated, the residue was dissolved in water and the resulting solution was applied to a cation exchange resin column (SPS BIO Sulfopropyl). The product was eluted with a concentration gradient of pyridine-acetate buffer pH 5.5 from 0.05 M (500 ml) to 0.3 M (500 ml). The fractions containing the desired product were combined, evaporated and the oily residue was evaporated twice more with water. The product was crystallized from 60 ml of i-propyl alcohol. The precipitate was filtered off, washed on the filter with isopropyl alcohol and dried. Yield 3.2 g (72%). Rf 0.5 (chloroform-methanol-32% acetic acid system 60:45:20). The purity of the product according to HPLC 95.1%. The compound structure was confirmed by ¹ H-NMR spectrum data. FIG. 1 shows the ¹ H NMR spectrum of N-tyrosyl-4-carbonyl-5-methyl-1,3-oxosolidine (Ia) in DMSO-d ₆ at 300K.

Example 2. Preparation of N-tyrosyl-4-carboxy-1,3-thiazolidine (Ib)

To the suspension, 5.6 g (42.0 mmol) of 4-carboxy-1,3-thiazolidine, prepared according to a known method [M.R. Vishnumaya, VK Singh Highly efficient reaction in organic and aqueous media // J.Org. Chem. 2009, v. 74, p. 4289-4297] in 100 ml of dry DMF with stirring was added 7.6 ml (54.0 mmol) of triethylamine and 20.1 g (42.0 mmol) of Boc ₂ -Tyr -ONSu. The reaction mixture was stirred at room temperature for 15 hours. The solvents were evaporated, the residue was dissolved in 150 ml of ethyl acetate and washed successively with 5% sulfuric acid solution and water until neutral pH, the organic layer was evaporated. The residue was dissolved in 50 ml of methylene chloride and 50 ml of trifluoroacetic acid was added. The reaction mixture was kept at room temperature for 1 hour and evaporated. The residue was dissolved in water and the resulting solution was applied to a cation exchange resin column (SPS BIO Sulfopropyl). The column was successively washed with two volumes of pyridine-acetate buffer pH 5.5, increasing the ionic strength of the buffer from 0.05 M to 0.3 M. The fractions containing the desired product were combined and evaporated. The residue was crystallized from 200 ml of isopropyl alcohol. The precipitate was filtered off, washed on the filter with isopropyl alcohol and dried. Yield 9.2 g (86.6%), Rf 0.5 (system chloroform-methanol-32% acetic acid -60: 45: 20). The purity of the product according to HPLC more than 95.3%. The compound structure was confirmed by ¹ H-NMR spectrum data. FIG. 2 shows the ¹ H NMR spectrum of N-tyrosyl-4-carboxy-1,3-thiazolidine (Ib) in DMSO-d ₆ at 300K.

¹ H NMR spectra were taken on a WM-500 spectrometer (Bruker) 500 MHz in DMSO-d ₆ at 300 K, the concentration of tyrosine derivatives was 2-3 mg / ml. Chemical shifts were measured relative to tetramethylsilane.

The study of biological activity was performed in accordance with regulatory documents.

All procedures in the study were performed in accordance with the approved Study Plan and Standard Operating Procedures (SOP).

Biological test system

The number of animals involved in the study was sufficient to fully register the effects studied and to statistically process the data obtained. The females were not pregnant or giving birth.

Type of animals, breed / line:

Mice - In ALB / c (males)

Rats - outbred (females)

Starting weight: Mice - 25-28 g. Rats - 210-260 g

Principles of selection of animals and distribution by groups: by weight and age by random selection. The body weight of each animal did not deviate from the average for the group by more than 20%.

All animal manipulations were carried out according to the rules adopted by the European Convention for the Protection of Vertebrate Animals used for research and other scientific purposes (European Conventionforthe Protectionof Vertebrate Animals Usedfor Experimentalandother Scientific Purposes (ETS 123) Strasbourg, 1986) and Directive 2010/63 / EU of the European Parliament and the Council of the European Union of September 22, 2010 on the protection of animals used for scientific purposes.

Experimental technique

The studied drugs - creams for external use (hereinafter referred to as "Cream A" and "Cream B")

Active ingredient - claimed tyrosine derivatives of formula (I)

Mice were put and rubbed into the foot 0.02 ml of the tested cosmetic, rats 0.1 ml. This is the maximum possible amount of cream that is well absorbed and does not remain on the skin surface. It was experimentally established that 1 ml of cosmetic weighs 1008 mg. For mice with an average body weight of 25 g, the dose was 20 mg per mouse or 800 mg / kg. For rats with an average body weight of 250 g, the dose was 101 mg or 404 mg / kg.

“Cream A” and “Cream B” were taken into a syringe with a volume of 1 ml and dosed out to mice and rats on an experienced paw, massaged in until the cream was completely absorbed.

Example 3. The study of the anti-inflammatory action of the proposed creams in the acute experiment when creating formalin edema

The acute inflammatory reaction was reproduced by subplantar introduction of a 2% formalin solution in a volume of 0.02 ml into the right hind paw.

Declare "Creams A and B" was applied on the right paw for 2 and 1 hour before the introduction of formalin. The control animals were rubbed into the right paw by a neutral cosmetic cream used as the basis for creating the tested cosmetic products (hereinafter referred to as the “Control Cream”).

After the observation, the animals were euthanized by cervical dislocation. Table 1 shows the conditions for the acute experiment.

As a result of subplantar administration of 0.02 ml of a 2% aqueous solution of formalin into the right hind paw, mice developed pronounced edema. The thickness of the paws was measured before the introduction of formalin, after 3 hours, 1 and 2 days after the induction of inflammation using the MCC-25 micrometer (OOO NPP ChIZ). The severity of edema was assessed as a percentage of the difference in thickness of the paw at the time of measurement and before formalin edema. Anti-inflammatory activity was calculated by the formula:

According to the data obtained, a comparative analysis was performed in the groups that received “Control cream”, “Cream A” and “Cream B”; the results of the effect of the inventive creams on the severity of “formalin” edema of the mouse paw (foot thickness, μm) are given in Table 2 and FIG. 3 shows a graph of the effect of the inventive creams on the severity of "formalin" edema of the mouse paw (% edema).

It was noted that in all periods of paw measurement, in mice treated with “Cream B”, the percentage of edema was less than in animals treated with “Control cream” and “Cream A”.

Example 4. The study of the analgesic action of the inventive creams in an acute experiment when creating formalin edema

As in Example 3, the acute inflammatory response was reproduced by subplantar administration of a 2% formalin solution in a volume of 0.02 ml into the right hind paw.

Declare "Creams A and B" was applied on the right paw for 2 and 1 hour before the introduction of formalin. The control animals in the right foot rubbed "Control cream".

After the observation, the animals were euthanized by cervical dislocation. The design of the study in the context of an acute experiment is similar to that in Example 3 (see Table 1).

During the first hour after the creation of formalin inflammation, each animal was observed, noting signs of pain. After 3 hours, 1 and 2 days, the mice were tested for motor activity, static-strength endurance and swimming duration. During these periods, the measurement of the affected limb was carried out and the condition of the animals was monitored. After the observation, the animals were euthanized by cervical dislocation.

Study of an analgesic action on animal behavior

After the introduction of formalin in mice during the first 60 minutes, the number of pain reactions in 4 types of behavior was taken into account:

0 - no reaction;

1 - the paw remains on the floor, but the animal does not rely on it;

2 - paw raised;

3 - the animal licks or bites its paw, or shakes or twitches it.

The criterion of analgesic effect was considered a significant decrease in the number of pain reactions in the groups that received the claimed "Cream A and B", relative to the control group.

Immediately after the subplantar administration of formalin, the mice showed great concern. After returning to the cage, they began to lick the injected paw hard. Within an hour, all mice did not rely on the affected limb. It is noticed that mice whose paws were treated with “Cream B” showed less concern than mice of the other two groups. Table 4 shows the number of lifting the paw and licking it, expressed in points. Table 3 presents data on the effect of the inventive creams on the pain threshold. FIG. 4 shows a graph of the effect of the tested creams on the pain threshold. From the data provided it is clear that the use of "Cream B" more than other creams reduces the intensity of the painful response to the introduction of formalin.

Study of the analgesic action of motor activity in the open field test

After 3, 24 and 48 hours after the creation of the formalin model of animals, one individual was placed in an experimental open field installation, which is a chamber of 330 × 430 × 205 mm. The floor of the camera is divided into 20 squares. For 2 minutes, the number of vertical posts and the number of crossed (4 legs) squares were taken into account. The criterion of analgesic action was considered an increase in horizontal and vertical activity. Data on the effect of the tested creams on the motor activity of mice are presented in table. 4. FIG. 5 also shows a graph of the effect of the inventive creams on the motor activity of mice. 3 hours after the introduction of formalin, all the mice moved actively around the arena and stood on their hind legs, relying on the affected limb. It is noted that animals whose experienced paws received cream “Cream B”, after 3 hours and 24 hours, were more active on their hind legs compared to the group that was administered “Control cream”. However, the best result is shown in the group whose paws were treated with gel "Cream A". In this group of mice more often crossed the squares and stood on the hind limbs.

Example 5. The study of the anti-inflammatory effect of "Cream" in a subchronic experiment in the creation of adjuvant arthritis.

Research methodology

Chronic inflammation (adjuvant arthritis) was modeled on female rats by subplantar administration of 0.1 ml of Freund's complete adjuvant (PAF) into the right hind paw (Becton, Dickinson and Company, U.S.A. Lot 6292862). The control cream and the claimed cosmetics were applied to the rats one day after the administration of PAF and then daily for 27 days. The inflammatory response was evaluated in dynamics every 2-3 days by the intensity of inflammatory changes in the joints of the affected limb, expressed as a percentage of edema, as well as by changes in pain sensitivity thresholds. The primary reaction (edema on the injection paw) was evaluated on the 3rd day after the injection of the adjuvant.

On the second day after the cancellation of “Crem”, animals were tested for the presence of withdrawal syndrome.

Table 5 shows the conditions for the subchronic experiment.

Statistical processing of the data was performed using Microsoft Office Excel 2010 software. The group arithmetic mean (M) and standard error of the mean (m) were calculated. Statistical significance of differences was assessed by Student's t-test. Reliable considered the difference between these groups at p <0.05.

The course of experimental adjuvant-induced arthritis was characterized by a change in various parameters. During the first 24 hours, hyperemia and edema of the hind right limb were visually observed in all rats (injection site of Freund's complete adjuvant). A change in the gait was also noted - the animals protected the injured limb, avoiding stepping on it, pressed their paw up. The analgesic effect of the tested creams was noted in the process of measuring the affected limb. It was established that in most animals of the groups that received the claimed cosmetic products - “Cream A” and “Cream B”, a weak analgesic effect occurred by 5 days of the experiment, in the control group of animals that received the “Control cream” cream - by 7 days. Further, the pain response in rats decreased with a decrease in the swelling of the affected limb.

The course of experimental adjuvant-induced arthritis was characterized by a change in various parameters. During the first 24 hours, hyperemia and edema of the hind right limb were visually observed in all rats (injection site of Freund's complete adjuvant). A change in the gait was also noted - the animals protected the injured limb, avoiding stepping on it, pressed their paw up. Over time, in the control animals, the edema in the right paw increased, and from the 12th day of the experiment another limb began to swell (secondary immunological reaction), the motor activity was reduced, the rats did not rely on the injured limb. Experienced animals that received the claimed cosmetic products were mobile, relied on all limbs, stood on their hind legs. The difference between the left and right paw was not noticeable.

The dynamics of the volume of the affected limb was evaluated by the thickness of the test foot, which was measured using a MCC-25 micrometer. The obtained data on the effect of the inventive creams on the dynamics of the thickness of the affected limb of rats are shown in Table 6. FIG. 6 shows the Dynamics of increase in the volume of the rat paws with adjuvant arthritis,%. Throughout the study, rats treated with “Cream B” had a lower edema percentage than rats treated with “Cream A” and “Control Cream”.

Example 6. Study of the development of withdrawal syndrome

In order to assess the possible withdrawal syndrome, an assessment was made of the main indicators of the behavior and reflexes of animals after the abolition of the prolonged application of the proposed creams.

The general condition of the animals on the second day after the cancellation of the creams did not change. When pressed with tweezers on the injured limb, all the rats were twitching it, when skin irritation of the external auditory canal using a paper tube was shaken with the head (ear reflex positive), when touching the hair cornea, they closed the eyelids (corneal reflex positive). When observing the rats, convulsions, tremors, jumps, shaking, teeth knocking, scratching, sneezing were not detected.

Data on the effect of the claimed creams on the emotional-motor activity of rats in the open-field test are presented in Table 7. After the application of the proposed creams was discontinued, the indicators of the emotional-physical activity of rats did not significantly differ between the groups.

After the cessation of the use of the claimed cosmetics "Cream A" and "Cream B", no withdrawal syndrome was detected.

Thus, an experimental study of the comparative pharmacological activity of cosmetics: “Control cream”, “Crema A” containing N-tyrosyl-4-carbonyl-5-methyl-1,3-oxozolidine and “Crema B” containing N-tyrosyl -4-carboxy-1,3-thiazolidine showed that the claimed creams have anti-inflammatory, analgesic and antiexudative action.

The most pronounced effect has "Cream B".

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Claims (3)

Heterocyclic tyrosine derivatives of the general formula

possessing anti-inflammatory, analgesic and anti-edematous action.

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